Association of High Sensitivity C-Reactive Protein with Type II Diabetic Retinopathy

Objective: The objective of this study is to find out the association of diabetic retinopathy with high sensitivity (hs) CRP levels.
Methodology:It was a descriptive cross-sectional study. The study was conducted at the Army Medical College's Department of Physiology and Centre for Research in Experimental and Applied Medicine (CREAM), Rawalpindi, in partnership with the Armed Forces Institute of Ophthalmology (AFIO), Rawalpindi for a tenure of 12 months. Ninety subjects were included in the study, thirty in each group: controls, diabetic subjects, and patients suffering from diabetic retinopathy (DR).
Results:Healthy controls, diabetic subjects, and patients with DR were found to have mean Fasting Plasma Glucose (FPG) values of 5.51 0.34 (mmol/l), 8.11+/- 0.67 (mmol/l), and 8.73+/- 0.90 (mmol/l), respectively (p=0.001). The mean HbA1c level in normal subjects was noted as 5.08 +/- 0.27 in healthy controls as compared to 7.70 +/-0.89 (mmol/l)) in diabetic subjects and 9.02+/-1.76 (mmol/l) in patients with DR (p=0.001 by ANOVA). In normal subjects, the mean hs CRP levels were noted to be 3.74+/-1.97 (mg/l) in healthy controls as compared to 15.32 +/- 2.93 (mg/l) in diabetic subjects and 26.71 +/- 4.88 (mg/l) in patients with diabetic retinopathy (p=0.001 by ANOVA).
Conclusion:During the study, it was found that elevated levels of these inflammatory biomarkers can accurately predict the onset of diabetic retinopathy and that hs CRP levels are strongly related to the development of diabetic retinopathy. Monitoring this inflammatory marker in the serum can therefore help prevent diabetic microangiopathic consequences, including DR.
Key words:Diabetic retinopathy, Fasting Plasma glucose, hs-CRP

Diabetes Mellitus (DM) is a worldwide health problem that is divided into two types with differences in the pathophysiology of type 1 and type DM.1 It is a silent killer which affects almost all body parts. Eyes, blood vessels, nerves and kidneys all are affected. The diagnosis of T2DM depends upon Plasma glucose level. The criteria to diagnose diabetes mellitus is the level of fasting plasma glucose ≥126 mg/dL (7.0mmol/l). The test for fasting plasma glucose is performed after not eating or drinking anything but water for 8 hours.2 Criteria for Random plasma Glucose (RBG) are 2-hour oral glucose tolerance test > 200 mg/dL (11.1mmol/l) and haemoglobin A1c ≥ 6.5%. If 2 hourly oral glucose tolerance test gives a result between 140mg/dl (7.8mmol/l) and 200mg/dl (11.1 mmol/l) then it is labelled as impaired glucose tolerance while levels of fasting plasma glucose 101mg/dl to 124mg/dl (5.6-6.9mmol/l) are considered as impaired fasting glucose (IFG).³ One of the most serious consequences in people with chronic hyperglycaemia is diabetic retinopathy (DR). Non-proliferative diabetic retinopathy (NPDR) and advanced-stage proliferative diabetic retinopathy (PDR) are the two stages of the disease. On clinical examination, signs of DR are micro-aneurysms, vitreous haemorrhage, retinal haemorrhage, exudates, cotton wool spots, neovascularization, and fibrosis. Diabetic Macular Edema (DME) is a condition in which the breakdown of the Blood Retinal Barrier (BRB) occurs which is responsible for fluid outflow and the proteins circulating into the neural retina.⁴
In 1930, the high-sensitivity C- C-reactive protein (hs-CRP) was discovered. It is an acute phase protein that hepatocytes produce in response to inflammation. hs-CRP possesses anti-inflammatory and pro-inflammatory properties. By binding to phospholipids, phosphocholine, chromatin, histone, and fibronectin, it serves an anti-inflammatory role in the identification and disposal of foreign particles and wounded cells.⁵
By triggering the traditional complement pathway and phagocytic cells via Fc receptors to hasten the removal of cellular waste, injured or apoptotic cells, and foreign particles, it causes inflammation. However, in ITP, hs-CRP can develop into a dangerous state when autoantibodies with the phosphocholine arm in auto-immune processes activate it. Through the activation of the complement system and inflammatory cytokines, it can occasionally make tissue injury worse.⁶
The hs-CRP levels respond to the onset and cessation of the inflammatory stimuli more accurately than the erythrocyte sedimentation rate (ESR), which is a proximate indicator of inflammation. Chronic inflammatory illness is indicated by persistently increased hs-CRP values. Hs CRP binds to phosphocholine, which is expressed on the surface of dead or damaged cells as well as some bacteria. This acts as a stimulant for the complement system, encouraging the phagocytosis of bacteria, necrotic, and apoptotic cells by macrophages. The term "acute phase response" refers to a rise in IL-6 concentrations produced by macrophages and adipocytes in response to a variety of acute and chronic inflammatory diseases such as bacterial, viral, or fungal infections.7Inflammation triggers the release of interleukin-6 and other cytokines, which stimulates hepatocytes to produce hs CRP and fibrinogen.
According to research, hs CRP levels are increased in both type 1 and type 2 diabetics. Many studies have found that high levels of hs CRP are linked to an increased risk of diabetic microvascular sequelae such as neuropathy, nephropathy, and retinopathy. Serum hs-CRP levels rise in diabetic retinopathy, although studies show that elevated serum hs-CRP levels are more common in patients with proliferative diabetic retinopathy than in those with non-proliferative diabetic retinopathy. According to research, serum hs CRP can be utilized as a biomarker of diabetic retinopathy.⁸ Blood flow to the retina is auto-regulated by means of many non-nervous mechanisms such as, endothelin-1. When the balance between endothelin-1 and another mediator such as nitric oxide (NO) is disturbed, it causes retinal hemodynamic impairment in diabetic retinopathy. Endothelin-1 has been implicated in the etiology of diabetic retinopathy, according to several studies. Furthermore, research points to the benefit of endothelin-1 antagonists in reducing diabetic complications. But the pathophysiology behind the association between high hs-CRP and diabetes sequelae, notably diabetic retinopathy, is still unknown.⁹ It has been believed that hs-CRP interacts with Fc receptors, specifically Fc receptor I (CD64) and Fc receptor II (CD32), to produce reactive oxygen species (ROS) and promote inflammation. As a result of hs-CRP's activation of the NF-B signalling pathway, pro-inflammatory molecules such as TNF and ROS are produced.10 Hence we designed the study to investigate the correlation between diabetic retinopathy and elevated hs CRP. This study will help the ophthalmologist in detecting means of early non-invasive diagnosis of diabetic retinopathy.

Healthy, diabetic, and diabetic retinopathic subjects were taken from the medical OPD of Pak Emirates Military Hospital (PEMH) and Armed Forces Institute of Ophthalmology, Rawalpindi (AFIO). Each group comprised of thirty subjects. Male, female, and their attendants visiting OPD from November 2019 to November 2020 were interviewed. Informed consent was taken from the subjects who were fulfilling the inclusion criteria and within the age ranges of 35-55 years. The subject’s details were taken including medical history. The initial classification of subjects was carried out through a preliminary examination.
AFIO and Army Medical College's ethical review committee gave their formal clearance before the study could be officially conducted. From all of the patients and healthy volunteers, informed consent was obtained in writing as per Performa attached as annexure –I and II. Type 2 diabetes without complications and diabetic retinopathy patients confirmed by Ophthalmologists were enrolled in the study.
After accomplishing their demographic details with history and clinical examination, Serum hs CRP levels were determined by drawing blood samples. Strict aseptic procedures were followed when drawing blood samples. Venepuncture was used to obtain 5ml of blood, of which 2.5ml was transferred to an EDTA tube for full blood analysis and the remaining 2.5ml was centrifuged for ten minutes at 2000-3000 revolutions per minute for separation of serum. For further examination at the CREAM laboratory, serum was pipetted out, transferred to Eppendorf tubes, and then stored at -80 c.

Healthy controls, diabetic subjects, and patients with DR were found to have mean Fasting Plasma Glucose (FPG) values of 5.51 0.34 (mmol/l), 8.11+/- 0.67 (mmol/l), and 8.73+/- 0.90 (mmol/l), respectively (p=0.001). The mean HbA1c level in normal subjects was noted as 5.08 +/- 0.27 in healthy controls as compared to 7.70 +/-0.89 (mmol/l)) in diabetic subjects and 9.02+/-1.76 (mmol/l) in patients with DR (p=0.001 by ANOVA). In normal subjects, the mean hs CRP levels were noted to be 3.74+/-1.97 (mg/l) in healthy controls as compared to 15.32 +/- 2.93 (mg/l) in diabetic subjects and 26.71 +/- 4.88 (mg/l) in patients with diabetic retinopathy (p=0.001 by ANOVA).
Table 1: Age, FBG, RBG, HbA1c, serum homocysteine, hs CRP, and BMI compared by one-way ANOVA between the normal healthy, diabetic, and diabetic retinopathic groups

The significance level is set at P 0.05 and all values have been reported as Mean + SD. FBG: Fasting Blood Glucose, RBG: Random Blood Glucose, HbA1c: Glycosylated haemoglobin, hs CRP: C - reactive protein, BMI: Body Mass Index TABLE 2: Comparison of age, FBG, RBG, HbA1c, serum homocysteine, hs CRP and BMI between two groups by post-hoc Tukey test

Table 3: Correlation of age, FBG, RBG, HbA1c, BMI with serum hs CRP

The BC-6200 Hematology Analyzer, which uses SF-Cube technology, is a noteworthy development in the field of diagnosing malaria. With the addition of clinically significant suspect flags, the three-dimensional cell analysis offered by SF-Cube technology permits comprehensive evaluations of white blood cells (WBC), reticulocytes (RET), and nucleated red blood cells (NRBC). 'Infected RBC' as a specialized flag is in line with the changing hematological analyzer market, where accurate and timely infectious agent identification is critical.
The capacity to differentiate between red blood cells that are infected plays a significant role in the timely and accurate identification of malaria, which is essential for better patient outcomes. The BC-6200's diagnostic skills are improved by the thorough examination of routine data, questionable flags, and the 'infected RBC' parameter. A comprehensive picture of the patient's hematological profile in relation to infection is provided by the interaction of established hematological parameters with indicators unique to malaria.¹³

Table 3: Blood Cell Parameters and Suspect Flags between Control Group and Malaria Groups in BC-6200 Analyzer

A study done by Katarzyna Kulik et al., concluded that RDW (r =.8350), MPV (r =.7634), Mon# (r =.8366), Baso# (r =.9205), and NRBC (r =.3768), the BC-6200 analyzer and ADVIA 2120i exhibited a strong association (r ≥.97). Compared to the ADVIA 2120i (r =.5677), the BC-6200 demonstrated a higher correlation with microscopic examination for NRBC (r =.8902). For flagging blasts (80.4%), immature granulocytes (80.5%), and abnormal lymphocytes (69.0%), the BC-6200 has demonstrated good efficiency.¹⁴
According to research by S. A. Khan et al., microscopy is the gold standard for identifying malaria. Even though it takes a long time—one test takes sixty minutes—if done by a patient and a qualified technologist, it is affordable and accurate. The Ideal exam, in contrast, is costlier but simpler and more objective. It also doesn't require any equipment. However, because there are fewer hospital admissions and morbidity, the cost element becomes less important.¹⁵
Another study by Germán Campuzano-Zuluaga et al., found that the identification of malaria using hematology analysers can be helpful as an adjuvant diagnostic tool in the work-up of feverish patients, as it is a by-product of its primary function, the CBC analysis. In order to quickly diagnose malaria and begin treatment, it would be ideal to include a flag for the disease and utilize it to direct microscopic examination of the patient's blood. Presently, the Coulter GEN·S, LH 750, and Sysmex XE-2100 analysers can be automated to detect malaria with the use of a Laboratory Information System. However, it is recommended that these numerical diagnostic criteria be verified using population-based samples. The industry's involvement is essential to these advancements, and it would be ideal if hematology analyzer manufacturers were willing to assess and incorporate algorithms in their devices that could identify samples that have a high probability of being malarial. This approach could help produce more precise algorithms in devices that are otherwise simpler.¹⁶ According to a comparison evaluation conducted by Katarzyna Kulik et al., the new Mindray BC-6200 analyzer offers good linearity and precision. With the ADVIA 2120i analyzer, morphological and 5-diff findings showed a very excellent correlation for the majority of evaluated parameters. Moreover, the BC-6200 outperformed the ADVIA 2120i in terms of correlation with microscopic evaluation in NRBC determination. A great deal of crucial information about the tested sample may be obtained by analyzing the scatter grams and flags produced by the BC-6200. This process also makes it easier to identify samples that need to be examined under a microscope.¹⁷ According to a study by G. da Rin1 et al., Mindray BC-6800, Sysmex XN-module, and Sysmex XE-2100 displayed low mean bias in Bland-Altman's plot that was comparable with LoD and LoQ, but differences between Siemens ADVIA 2120i and Coulter UniCel DxH 800 were higher and increased proportionately with increasing NRBC concentration. It is significant to note that each of the two skilled hematologist counted 1000 cells at 400× magnification in order to improve the accuracy of the manual NRBC counts.¹⁸
According to a study by Tae Hwan Lee, M.D. et al., the BC-6200 performed exceptionally well when it came to background, carryover, and precision CBC parameter results. Moreover, there was a strong correlation between the CBC values and the standard instrument (XE-2100). The flagging's efficiency, specificity, and sensitivity were also deemed satisfactory. We come to the conclusion that BC-6200 is a capable HA that can fulfil the demands of mid-volume testing in clinical laboratories and deliver trustworthy and accurate diagnostic results.¹⁹

The study concludes by highlighting the importance of the 'infected RBC' flag and malaria-specific parameters, as well as the usefulness of the BC-6200 Hematology Analyzer in the diagnosis of malaria. This invention supports further efforts to improve infectious disease diagnostic accuracy, especially in areas where malaria represents a substantial health burden. The BC-6200 is a potentially useful weapon in the front-line fight against malaria and other blood-borne infections as technology develops. However, because to their tiny size and poor nucleic acid content, the ring stage and early trophozoite stage of Plasmodium cannot be readily detected on BC-6200.

1. Talapko J, Škrlec I, Alebić T, Jukić M, Včev A. Malaria: the past and the present. Microorganisms. 2019 Jun 21;7(6):179.; https://doi.org/10.3390/microorganisms7060179
2. Luepke KH, Suda KJ, Boucher H, Russo RL, Bonney MW, Hunt TD, et al. Past, present, and future of antibacterial economics: increasing bacterial resistance, limited antibiotic pipeline, and societal implications. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy 2017;37(1):71-84. https://doi.org/10.1002/phar.1868
3. Ningombam A, Sarkar A, Acharya S, Chopra A, Kumar K, Subramanian A. Application of Sysmex XN-series automated haematology analyser in the rapid detection of malaria. Indian Journal of Hematology and Blood Transfusion. 2020 Jul;36:512-8. https://doi.org/10.1007/s12288-020-01276-x
4. Sori G, Zewdie O, Tadele G, Samuel A. External quality assessment of malaria microscopy diagnosis in selected health facilities in Western Oromia, Ethiopia. Malar J.2018;17:233. https://doi.org/10.1186/s12936-018-2386-2.
5. Sun Y, Xiang D, Chen C, He S, Qi H, Wang C. Infected RBC flag/parameter provided by Mindray BC-6800 haematology analyzer aid the diagnosis of malaria. Malaria Journal. 2019 Dec;18(1):1-8. https://doi.org/10.1186/s12936-019-2890-z
6. Silamut K, Phu NH, Whitty C, Turner GD, Louwrier K, Mai NT, et al. A quantitative analysis of the microvascular sequestration of malaria parasites in the human brain. 1999;155(2):395-410.
7. Anselmo FC, Soumanou AG, de Aguiar Ferreira C, Sobrinha FM, Castro AC, Brito RO, Mota AJ, de Souza Gonçalves M, Neto JP. THE HEMATOLOGICAL PARAMETERS AND BIOCHEMICAL MARKERS OF IRON STATUS IN ALFA-THALASSEMIA 3.7 KB DELETION FROM METROPOLITAN REGION OF MANAUS, AMAZONAS, BRAZIL.: alfa-Thalassemia 3.7 deletion From Amazonas, Brazil. Mediterranean Journal of Hematology and Infectious Diseases. 2021 Jan 1;13(1):e2021001-.
8. Gerke O. Reporting standards for a Bland–Altman agreement analysis: A review of methodological reviews. Diagnostics. 2020 May 22;10(5):334. https://doi.org/10.3390/diagnostics10050334
9. Khan SA, Anwar M, Hussain S, Qureshi AH, Ahmad M, Afzal S. Comparison of Optimal malarial test with light microscopy for the diagnosis of malaria. JOURNAL-PAKISTAN MEDICAL ASSOCIATION. 2004 Aug 1;54(8):404-7.
10. Kulik K, Kwiecień I, Chełstowska B, Rutkowska E, Rzepecki P. Evaluation and comparison of the new Mindray BC‐6200 hematology analyzer with ADVIA 2120i. International Journal of Laboratory Hematology. 2021 Jun;43(3):395-402.https://doi.org/10.1111/ijlh.13418
11. Lee TH, Kim H, Park M, Hur M, Lee CH. Performance Evaluation of the Mindray BC-6200 Hematology Analyzer; Comparison with Sysmex XE-2100 and Manual Microscopy. Laboratory Medicine Online.;12(4):269-77.DOI: https://doi.org/10.47429/lmo.2022.12.4.269